IL-6 promotes M2 macrophage polarization by modulating purinergic signaling and regulates the lethal release of nitric oxide during Trypanosoma cruzi infection

The production of nitric oxide (NO) is a key defense mechanism against intracellular pathogens but it must be tightly controlled in order to avoid excessive detrimental oxidative stress. In this study we described a novel mechanism through which interleukin (IL)-6 mediates the regulation of NO release induced in response to Trypanosoma cruzi infection. Using a murine model of Chagas disease, we found that, in contrast to C57BL/6 wild type (WT) mice, IL-6-deficient (IL6KO) mice exhibited a dramatic increase in plasma NO levels concomitant with a significantly higher amount of circulating IL-1β and inflammatory monocytes. Studies on mouse macrophages and human monocytes, revealed that IL-6 decreased LPS-induced NO production but this effect was abrogated in the presence of anti-IL-1β and in macrophages deficient in the NLRP3 inflammasome. In accordance, while infected WT myocardium exhibited an early shift from microbicidal/M1 to anti-inflammatory/M2 macrophage phenotype, IL6KO cardiac tissue never displayed a dominant M2 macrophage profile that correlated with decreased expression of ATP metabolic machinery and a lower cardiac parasite burden. The deleterious effects of high NO production-induced oxidative stress were evidenced by enhanced cardiac malondialdehyde levels, myocardial cell death and mortality. The survival rate was improved by the treatment of IL-6-deficient mice with a NO production-specific inhibitor. Our data revealed that IL-6 regulates the excessive release of NO through IL-1β inhibition and determines the establishment of an M2 macrophage profile within infected heart tissue.Fil: Sanmarco, Liliana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ponce, Nicolás Eric. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Visconti, Laura M.. Hospital Nuestra Señora de la Misericordia; ArgentinaFil: Eberhardt, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Theumer, Martín Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Minguez, Ángel R.. Hospital Nuestra Señora de la Misericordia; ArgentinaFil: Aoki, Maria del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL-DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome

Identification of astrocyte regulators by nucleic acid cytometry

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers

Gut-licensed IFNγ+ NK cells drive LAMP1+TRAIL+ anti-inflammatory astrocytes

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR–Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL–DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome.Fil: Sanmarco, Liliana Maria. Harvard Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wheeler, Michael A.. Harvard Medical School; Estados UnidosFil: Gutiérrez Vázquez, Cristina. Harvard Medical School; Estados UnidosFil: Polonio Manganeli, Carolina. Harvard Medical School; Estados UnidosFil: Linnerbauer, Mathias. Harvard Medical School; Estados UnidosFil: Pinho Ribeiro, Felipe A.. Harvard Medical School; Estados UnidosFil: Li, Zhaorong. Harvard Medical School; Estados UnidosFil: Giovannoni, Federico. Harvard Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Batterman, Katelyn V.. University Of Boston. School Of Medicine.; Estados UnidosFil: Scalisi, Giulia. Harvard Medical School; Estados UnidosFil: Zandee, Stephanie E. J.. University of Montreal; CanadáFil: Heck, Evelyn Sabrina. Harvard Medical School; Estados Unidos. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alsuwailm, Moneera. Harvard Medical School; Estados UnidosFil: Rosene, Douglas L.. University Of Boston. School Of Medicine.; Estados UnidosFil: Becher, Burkhard. Universitat Zurich; SuizaFil: Chiu, Isaac M.. Harvard Medical School; Estados UnidosFil: Prat, Alexandre. University of Montreal; CanadáFil: Quintana, Francisco Javier. Harvard Medical School; Estados Unido

MAFG-driven astrocytes promote CNS inflammation

Multiple sclerosis is a chronic inflammatory disease of the CNS$^{1}$. Astrocytes contribute to the pathogenesis of multiple sclerosis$^{2}$, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis

Droplet-based forward genetic screening of astrocyte–microglia cross-talk

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms

AHR is a Zika virus host factor and a candidate target for antiviral therapy

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity

Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells 1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders 3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α–NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activationThis work was supported by grants NS102807, ES02530, ES029136 and AI126880 from the National Institutes of Health (NIH); RG4111A1 and JF2161-A-5 from the National Multiple Sclerosis Society; RSG-14-198-01-LIB from the American Cancer Society; and PA-160408459 from the International Progressive MS Alliance (to F.J.Q.). C.M.P. was supported by a fellowship from FAPESP BEPE (2019/13731-0) and by the Herbert R. & Jeanne C. Mayer Foundation; G.F.L. received support from a grant from the Swedish Research Council (2021-06735); C.G.-V. was supported by an Alfonso Martin Escudero Foundation postdoctoral fellowship and by a postdoctoral fellowship (ALTF 610-2017) from the European Molecular Biology Organization; C.-C.C. received support from a postdoctoral research abroad program (104-2917-I-564-024) from the Ministry of Science and Technology, Taiwan; C.M.R.-G. was supported by a predoctoral F.P.U. fellowship from the Ministry of Economy and Competitiveness and by the European Union FEDERER program; M.A.W. was supported by NIH (1K99NS114111, F32NS101790 and T32CA207201), the Program in Interdisciplinary Neuroscience and the Women’s Brain Initiative at Brigham and Women’s Hospital; T.I. was supported by an EMBO postdoctoral fellowship (ALTF: 1009–2021) and H.-G.L. was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A3A14039088). We thank L. Glimcher and J. R. Cubillos Ruiz for sharing ItgaxXbp1 mice; S. McSorley for providing the S. typhimurium strain; H. Xu and M. Lehtinen for providing training on CSF extraction; all members of the F.J.Q. laboratory for advice and discussions; R. Krishnan for technical assistance with flow cytometry studies; and the NeuroTechnology Studio at Brigham and Women’s Hospital for providing access to Seahorse instruments. Cre f lo

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets